Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples.
Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples.
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DOI：
10.3390/ijms16023537
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发表时间：
2015-02-05
影响因子：
5.6
通讯作者：
Hummon AB
中科院分区：
文献类型：
--
作者：
Feist P;Hummon AB
蛋白质调节许多细胞功能，分析生物样品中蛋白质的存在和丰度是蛋白质组学的中心焦点。发现和验证各种疾病的生物标志物，途径和药物靶点可以使用基于质谱的蛋白质组学来完成。然而，对于像肿瘤活检这样的质量有限的样品，获得足够量的蛋白质以生成高质量的质谱数据可能是具有挑战性的。为大规模量开发的技术从毫克量的起始材料中回收了足够量的蛋白质，但是当只有微克量的材料可用时，这些技术的样品损失变得严重。为了应对这一挑战，蛋白质组学家开发了微型技术，这些技术与减少的样本量（100 μg或更低）兼容，但仍然能够实现出色的蛋白质组覆盖率。提取，污染物去除，蛋白质定量，和微克蛋白质范围内的样品处理技术进行审查，重点是液相色谱和自下而上的质谱兼容技术。此外，一系列的生物标本，包括哺乳动物组织和模型细胞培养系统，进行了讨论。
Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.
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